Figure 2

LBS 4D multicolor imaging.

(a) The illumination geometry of LBS, where the light sheet is tilted 30° to the surface of coverslip. Scanning is done by horizontal movement of the coverslip while keeping the light sheet fixed. Fixed HT22 cells double-stained with Alexa488-tagged anti-tubulin (Green) and Alexa555-tagged anti-Tom20 (Red and antigen in the mitochondria outer membrane) were imaged by different light sheet configurations in the same region. (b) Single slices in a “raw” 3D stack (before de-skewing). In the image obtained by Gaussian light-sheet, the SNR reduced rapidly from the bottom (where the waist of Gaussian light-sheet is aligned) to the top due to the rapid beam diffraction. On the other hand, LBS1 produced optical sections with minimal background and high SNR across the entire field of view. LBS2 exhibits multiple peaks in the axial point spread function, but can be sharpened after deconvolution. (c,d) show the two-color 3D rendering of HT22 cells (tubulin in green and Tom20 in red) scanned with LBS1 and LBS2 with deconvolution, respectively. From left to right: the two color 3D rendering of the cell; a zoom-in of the boxed region; the intensity plot of the tubulin signal along the doted yellow line. (e) 4D multicolor imaging of live tobacco BY-2 cells using LBS1. Green puncta represent the AtSCAMP3-GFP labeled clathrin coated endocytic vesicles, while the red fluorescent FM4-64 dye labels all the endocytic vesicles derived from plasma membrane. The 3D distributions of both types of vesicles are clearly resolved in time and space. From left to right: the 3D rendering of the first time point; the z projection and y projection at the boxed region at different time (the 17 sec and 35 sec), showing the change of vesicle distribution; and the axial profile along a vesicle marked by dotted line, showing the co-localization of two proteins. Scale bars: 5 μm.